Human monocytotropic ehrlichiosis (HME) is the most prevalent emerging tick-borne infectious disease caused by Ehrlichia chaffeensis, Gram negative obligate intracellular bacteria. The proposed studies will utilize two specific Ehrlichia strains;Ehrlichia muris, which causes persistent infection, and Ixodes ovatus Ehrlichia (IOE), which causes either lethal or sub-lethal infection, depending on the dose and route of inoculation. These models faithfully recapitulate mild and severe HME. Our studies have revealed that mice persistently infected with E. muris, but not mice that lack persistent infection or mice infected with the sub-lethal IOE infection that fails to establish persistent infection, are indeed protected against heterologous re-challenge with ordinarily lethal dose of IOE. Heterologous protection is mediated primarily by CD4+ type-1 memory responses and antigen specific IgG antibody response, while fatal primary and secondary ehrlichiosis is due to an extensive tissue injury mediated by Ehrlichia specific CD8+ T cells. Our preliminary data reveal that persistent E. muris infection is associated with substantial expansion of T regulatory cells, increased TGF-beta production in the spleens, and absence of immune mediated pathology following infection with IOE. These data indicate that we lack the fundamental understanding of the regulatory mechanisms that control the induction and maintenance of protective T cell memory and prevent the development of immunopathology upon secondary challenge, which is a prerequisite to developing an effective vaccine against HME. We will address this specific gap in our knowledge in the following two specific aims. In specific aim 1, we will determine the magnitude, phenotype, and function of memory T cells maintained in the presence or absence of persistent ehrlichial infection in C57BL/6 mice. Specific aim 2 will determine the role of T regulatory cells and TGF-beta in the persistence of ehrlichial infections, prevention of organ injury, and maintenance of protective memory T cell responses. The experiments utilize state-of-the art techniques such as multiparamter flow cytometry. Our study is significant as it address the strong need for balancing protective immunity and immunopathology in designing effective vaccination strategies. Knowledge generated therein will benefit vaccine development efforts not only against Ehrlichia but also, against other intracellular pathogens that are controlled by cell mediated immunity, which will significantly have major health impact. PUBLIC HEALTH RELEVANCE: Human monocytic ehrlichiosis is a major public health problem in many parts of the United States, due in part to the lack of a vaccine and inadequate therapy. The development of a vaccine for HME is lacking due to limited understanding of how to stimulate the memory T cells required for vaccine induced immunity. This proposal will determine how memory T cells are generated, maintained and regulated using a unique experimental animal model of the disease. The results from these experiments will provide a solid foundation of knowledge for the timely development of an effective and safe vaccine against ehrlichiosis.